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Acid-resistant saccharomyces cerevisiae and application thereof

A Saccharomyces cerevisiae, tolerance technology, applied in the field of microorganisms, can solve problems such as environmental pollution, neutralizer cost increase, increase separation and purification processes, etc., and achieve good growth, strong tolerance, and stable properties

Active Publication Date: 2020-09-18
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the above-mentioned low pH that inhibits the growth of host cells, the addition of neutralizers increases costs, increases subsequent separation and purification processes, and easily causes environmental pollution, etc.
In the present invention, in order to obtain cost-effective non-neutralizing fermentation host cells (cost-effective non-neutralizing fermentation host strains) that are not added during the fermentation process, malic acid is added exogenously as a screening pressure, and Saccharomyces cerevisiae is iteratively evolved to solve the problem of Saccharomyces cerevisiae low The problem of growth and production of short-chain organic acids under pH conditions, the main purpose of the invention is to improve the acid-resistant ability of Saccharomyces cerevisiae, and build a Saccharomyces cerevisiae acid-resistant chassis cell factory for the production of various short-chain organic acids

Method used

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  • Acid-resistant saccharomyces cerevisiae and application thereof
  • Acid-resistant saccharomyces cerevisiae and application thereof
  • Acid-resistant saccharomyces cerevisiae and application thereof

Examples

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Effect test

Embodiment 1

[0026] Example 1: Evolution and screening of acid-tolerant Saccharomyces cerevisiae cells

[0027] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, different concentrations of malic acid were added exogenously, and directed evolution screened to obtain Saccharomyces cerevisiae mutant strains that tolerated lower pH. In the present invention, by adding different concentrations of malic acid, the pH is controlled to decrease step by step (from pH 6.0, the addition of exogenous malic acid is 4.6g / L). Culture for enrichment (the target is the OD of the cells at 48h at this pH 600Stability is reached, and there is no significant increase in the first two times; the purpose is to increase the number of mutant cells and make cell growth more stable at this pH) before proceeding to the evolution of the next level of pH. Among them, the pH span of each stage from initial pH 6.0 to pH 4.0 is 0.2, namely 6.0, 5.8, 5.6...4.2, 4.0; the pH span of each stage from pH 4.0 to...

Embodiment 2

[0031] Example 2: Analysis of acid-resistant Saccharomyces cerevisiae cell traits

[0032] In order to verify the stability of the obtained S.cerevisiae mutant strain MTPfo-4 under low pH conditions, the growth of MTPfo-4 was compared with the starting strain CEN.PK2-1C under the condition of different amounts of exogenous malic acid. The added concentration (g / L) of exogenous malic acid is 0, 30, 40, 50, 60, 70, 80, 90, 100 respectively, and samples are taken every 12 hours until 72 hours, and the growth curve of the bacterial strain is measured, and the results are as follows: figure 2 As shown, compared with the starting strain CEN.PK2-1C, the mutant strain MTPfo-4 had significantly improved growth under the condition of exogenous malic acid addition, and had a strong tolerance to exogenous malic acid addition.

Embodiment 3

[0033] Example 3: Analysis of the ability of acid-tolerant Saccharomyces cerevisiae cells to tolerate other acids

[0034] In order to analyze the tolerance ability of the mutant strain MTPfo-4 to other acids, 10 kinds of acids were added through exogenous sources in the present invention, including 2 kinds of inorganic acids HCl, H 3 PO 4 , 8 kinds of organic acids lactic acid, malic acid, fumaric acid, succinic acid, tartaric acid, furoic acid, gluconic acid, citric acid, to analyze the acid resistance of cells, control the initial pH 2.44, take samples every 12h, until 72h, by measuring OD 600 To analyze the acid resistance of cells. The result is as image 3 As shown, compared with the starting strain CEN.PK2-1C, MTPfo-4 had no effect on 6 acids HCl, H 3 PO 4 , lactic acid, malic acid, citric acid, and gluconic acid were strongly tolerated, however, all 10 acids strongly inhibited the growth of CEN.PK2-1C under this pH condition. Because under the condition of pH 2.44...

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Abstract

The invention discloses an acid-resistant saccharomyces cerevisiae and application thereof. The saccharomyces cerevisiae MTPfo-4 disclosed by the invention is preserved in China Center for Type Culture Collection on June 10, 2020, and the preservation number is CCTCC M 2020199. According to the invention, exogenous malic acid is used as stress pressure, the S. Cerevisiae acid-resistant mutant strain MTPfo-4 is obtained through directed evolution screening in a laboratory, and the minimum tolerance pH is 2.44 which is also the minimum tolerance pH of currently reported saccharomyces cerevisiae.The mutant strain MTPfo-4 which is tolerant to various organic acids at the same time has the advantage that the tolerance to exogenous malic acid is increased to 86.8 g / L. The obtained mutant strainMTPfo-4 is further identified, is stable in character and good in growth, and can tolerate various organic acids (lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid andtartaric acid) at the same time, also has strong tolerance to inorganic acids (HCl and H2PO3), which is difficult to achieve in current S. Cerevisiae research reports. The acid-resistant saccharomycescerevisiae is expected to be used for producing various short-chain organic acids in an acid-resistant chassis cell factory.

Description

technical field [0001] The invention relates to an acid-resistant Saccharomyces cerevisiae and its application, belonging to the technical field of microbes. Background technique [0002] Short-chain organic acids, as natural metabolites of microorganisms, have been widely used in industries such as food, biomedicine, cosmetics, detergents, polymers and textiles in recent years. Short-chain organic acids produced by microorganisms mainly include propionic acid, pyruvate, lactic acid, 3-hydroxypropionic acid, tricarboxylic acid (TCA) cycle intermediate metabolite α-ketoglutarate, malic acid, succinic acid, fumaric acid and lemon acid. Among them, the calcium salt, sodium salt and ammonium salt of propionic acid have a strong antiseptic ability and can be used as a preservative in animal feed and human food. Itaconic acid can be used to prepare synthetic fibers, synthetic resins and plastics, and its ester derivatives can be used as plasticizers for styrene or polyvinyl chlo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/18C12P7/40C12P7/42C12P7/56C12P7/46C12P7/48C12P7/58C12P17/04C12R1/865
CPCC12N1/18C12P7/40C12P7/42C12P7/56C12P7/46C12P7/48C12P7/58C12P17/04C12R2001/865C12N1/185C12N1/36C12N1/14
Inventor 刘龙陈坚堵国成李江华吕雪芹孙利
Owner JIANGNAN UNIV
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